EP3897569A1

EP3897569A1 - Composition comprising a lactobacillus rhamnosus extract

Info

Publication number: EP3897569A1
Application number: EP19835377.3A
Authority: EP
Inventors: Marc Dumas; Catherine Heusele; Valérie GORZELANCZYK; Olivier JEANNETON
Original assignee: LVMH Recherche GIE
Current assignee: LVMH Recherche GIE
Priority date: 2018-12-21
Filing date: 2019-12-18
Publication date: 2021-10-27
Also published as: JP2022514669A; FR3090383B1; WO2020127581A1; KR20210116494A; CN113453660B; CN113453660A; FR3090383A1; US12011501B2; US20220054401A1

Abstract

The present invention concerns a composition for topical application to keratinous material, in particular the skin and/or lips, comprising, in a physiologically acceptable medium, at least one Lactobacillus rhamnosus extract (CNCM I-5313), one of its fractions and/or one of its metabolites and at least one excipient chosen from solvents other than water, UV filters, surfactants, gelling agents, thickeners, fillers, dyes, film-forming polymers, perfumes and the mixtures of same; and the uses thereof, in particular for promoting and/or improving the skin barrier function.

Description

Composition comprising an extract of Lactobacillus rhamnosus

FIELD OF THE INVENTION

The present invention relates to a composition comprising a new extract of Lactobacillus rhamnosus, in particular a lysate of Lactobacillus rhamnosus (CNCM 1-5313), and its beneficial effects on keratin materials, in particular the skin.

STATE OF THE ART

The skin is the first barrier of the human body. It protects the organs from differences in temperature, humidity, and aggressions from the external environment, such as UV rays, stress, pollution, pathogenic microorganisms ... However, excessive chemical and physical stimulations deteriorate the functions normal skin and induce or accelerate its aging, resulting in the appearance of wrinkles and a loss of firmness, flexibility and elasticity.

The search for new molecules or active ingredients usable in cosmetics is a necessity in order to be able to develop effective products to give a younger appearance to the skin, a homogeneous complexion, without colon irregularity at the level of pigmentation or microcirculation, a radiant complexion .

The Applicant has rightly demonstrated that an extract of Lactobacillus rhamnosus, in particular a lysate of Lactobacillus rhamnosus (CNCM 1-5313), had beneficial effects on skin cell cultures in vitro: it demonstrated that this lysate had a stimulating effect on the expression of several genes involved in particular in:

- detoxification (MRP1 = ABCC1, LONP1, GPX1),

- the functioning of the respiratory chain of the mitochondria involved in the production of cellular energy (MT = NADH dehydrogenase / ubiquinone),

- the secretion of hyaluronic acid (MRP5),

- maintaining the integrity of cellular structures such as that of the cell nucleus (SYNE2, LM NA), and

- the formation of several collagens and elastin (COL1A1, COL5A1, COL13A1,

ELN).

Thus, the extract of Lactobacillus rhamnosus according to the invention and in particular a lysate of Lactobacillus rhamnosus (CNCM 1-5313), is advantageous in that it has a detoxifying and antioxidant effect, an antiaging effect aimed at reducing wrinkles, of the loss of firmness and density (collagens and elastin and anchoring points between cells and these structural proteins), a strengthening effect on the skin (cellular structures like the nucleus of cells), an effect of adaptation of the skin to stress hypoxic, a depigmenting effect on skin spots (inhibition of melanogenesis) and / or a normalization effect of the skin microbiota via the strengthening of innate immunity and the skin barrier.

STATEMENT OF THE INVENTION

A first subject of the invention relates to a composition for topical application to keratin materials, in particular the skin and / or the lips, comprising, in a physiologically acceptable medium, at least one extract of Lactobacillus rhamnosus (CNCM I-5313), one of its fractions and / or one of its metabolites and at least one excipient chosen from solvents other than water, UV filters, surfactants, gelling agents, thickeners, fillers, materials coloring agents, film-forming polymers, perfumes and their mixtures, in particular solvents other than water, surfactants, gelling agents, thickeners, fillers, coloring matters, film-forming polymers, perfumes and their mixtures.

The invention also relates to a non-therapeutic cosmetic process comprising the application to keratin materials of at least one extract of Lactobacillus rhamnosus (CNCM 1-5313) according to the invention or of a composition containing it, intended to prevent and / or reduce the signs of skin aging and / or promote and / or improve the barrier function. We will talk indifferently about Lactobacillus rhamnosus (CNCM 1-5313) or Lactobacillus rhamnosus CNCM 1-5313, corresponding to the strain of Lactobacillus rhamnosus deposited according to the Budapest Treaty with the National Collection of Cultures of Microorganisms (CNCM) at the 'Institut Pasteur (28 rue du Docteur Roux, F-75024 Paris cedex 15) on April 1, 2018 on behalf of BIOVITIS, Le Bourg, 15400 SAINT-ETIENNE-DE- CHOMEIL, under the designation under the number CNCM 1-5313.

Another subject of the invention relates to the non-therapeutic cosmetic use of an extract of Lactobacillus rhamnosus (CNCM 1-5313) to promote and / or improve the barrier function and / or reduce the signs of skin aging.

DETAILED DESCRIPTION OF THE INVENTION

The present invention therefore relates in particular to a composition for topical application to keratin materials, in particular the skin and / or the lips, comprising, in a

RECTIFIED SHEET (RULE 91) ISA / EP physiologically acceptable medium, at least one extract of Lactobacillus rhamnosus (CNCM 1-5313), one of its fractions and / or one of its metabolites and at least one excipient chosen from solvents other than water, surfactants -actives, gelling agents, thickeners, fillers, coloring matters, film-forming polymers, perfumes and their mixtures.

By "keratin materials" is meant according to the invention the skin, the mucous membranes (in particular the lips), and the integuments. According to a particular embodiment, the composition is intended for topical application to the skin, the lips or the integuments, preferably the skin.

The “keratin materials” according to the invention are generally healthy keratin materials, that is to say that do not present disorders or disorders which would relate to a pathological state (“unhealthy” subjects, suffering from a pathology ). We will talk about healthy skin and / or lips or skin and / or lips in the rest of the description.

By "physiologically acceptable medium" is meant a medium compatible with keratin materials, in particular the skin and / or the lips.

The term “excipient” according to the invention means a substance devoid of cosmetic effect as such, but useful for incorporating Lactobacillus rhamnosus extract into the composition of the invention or for shaping this one.

Lactobacillus rhamnosus extract

According to the definitions used by the international scientific associations for probiotics and prebiotics, "probiotics" means living microorganisms which, when administered in an adequate quantity, have beneficial effects on the health of the host. The term “prebiotics” is understood to mean digestion-resistant food ingredients which induce specific changes in the composition and / or activity of the intestinal microbiota, thereby producing a beneficial effect on the health of the host. By "symbiotics" is meant products that contain both probiotics and prebiotics.

The extract of Lactobacillus rhamnosus (probiotic microorganism) according to the invention can be included in a composition according to the invention in a living, semi-active or inactivated, dead form. It can be included as fractions of cellular components or as metabolites. The microorganism, metabolite (s) or fraction (s) little (Fri) t also be introduced in the form of a lyophilized powder, a culture supernatant and / or if necessary in a concentrated form.

According to a particular embodiment, the microorganism Lactobacillus rhamnosus is used in an inactivated or even dead form, and more particularly in the form of a lysate.

Thus, according to a particular and preferred mode, the extract of Lactobacillus rhamnosus according to the invention is a lysate of Lactobacillus rhamnosus (CNCM 1-5313) (not living), of one of its fractions and / or of one of its metabolites.

We will talk indifferently in the description of prob probiotic extract ’or‘ Lactobacillus rhamnosus extract ’or prob probiotic extract’ or act Lactobacillus rhamnosus ’extract. By ‘lysat ’is meant in particular a material obtained by cell lysis of the cells of the microorganism considered, thus causing the release of the intracellular biological constituents naturally contained in said cells. The lysate used is formed in whole or in part of the intracellular biological constituents and of the constituents of the cell walls and membranes. The present invention therefore relates to the use of a probiotic lysate and / or one of its fractions and / or one of its metabolites.

Within the meaning of the invention, the term "fraction" more particularly designates a fragment of said microorganism endowed with the same beneficial effects on the skin, at least in part, by analogy to said whole microorganism.

In particular, the lysate according to the invention contains the cellular cytoplasmic fraction containing the enzymes (ex: dehydrogenases, phosphatases ...) and / or the constituents of the cell walls (ex: peptidoglycans ...) and / or the constituents of the membranes cells (ex: glycerophospholipids ...).

Cell lysis can be carried out by various technologies well known to those skilled in the art, such as for example osmotic shock, thermal shock, by ultrasound, or even under mechanical stress (eg centrifugation).

According to a preferred embodiment, the lysate is obtained by centrifugation and osmotic shock (ex: addition of soda).

According to a particular mode, the microorganism is cultured anaerobically in a suitable culture medium. When the stationary phase of development is reached, the culture medium can be inactivated by pasteurization, for example at a temperature of 60 to 65 ° C. for 30 min. The microorganisms are then collected by a conventional separation technique, for example membrane filtration, centrifugation and resuspended in a sterile physiological NaCl solution. The lysate can be obtained by ultrasonic disintegration of such a medium in order to release the cytoplasmic fractions, the cell wall fragments and the products derived from metabolism. Then all the components in their natural distribution are then stabilized in a weakly acidic aqueous solution.

The lysate thus obtained generally has a concentration of the order of 0.1 to 10%, in particular from 0.5 to 5% and in particular from 1 to 2% by weight in dry matter (s) (active ( s)) in relation to its total weight.

The lysate can be used in different forms, in the form of a solution or in the form of a powder.

According to a particular embodiment, the lysate is in the form of a solution, preferably an aqueous solution, comprising from 0.1 to 10%, in particular from 0.5 to 5% and in particular from 1 to 2% in weight of dry matter (s) (active) relative to its total weight.

The probiotic microorganism according to the invention belongs to the species Lactobacillus rhamnosus (ATCC 7469, CCM 1825, NCDO 243, NCIB 6375 or WDCM 00101 depending on the collections in which it has been deposited), preferably the strain Lactobacillus rhamnosus CNCM 1- 5313, filed according to the Budapest Treaty with the National Collection of Cultures of Microorganisms (CNCM) at the Institut Pasteur (28 rue du Docteur Roux, F-75024 Paris cedex 15) on April 1, 2018 in the name of BIOVITIS , Le Bourg, 15400 SAINT-ETIENNE-DE-CHOMEIL, under the designation CNCM 1-5313, and / or one of its fractions and / or one of its metabolites.

A lysate of this L. rhamnosus strain is obtained according to the following protocol from the supplier Greentech:

1. Culture of the L. rhamnosus strain in an appropriate culture medium;

2. Biomass recovery by centrifugation;

3. Cell lysis (addition of soda);

4. Adjustment of the pH (addition of citric acid) and dry matter (addition of sterile water) to obtain an active ingredient content ranging from 0.1 to 10% by weight of dry matter (active ingredient) relative to the total weight of said lysate;

5. Addition of preservative (phenoxyethanol).

Such a L. rhamnosus lysate is called Probiophyte LB by the supplier Greentech, and defined in INCI name under the name WATER and LACTOBACILLUS FERMENT

RECTIFIED SHEET (RULE 91) ISA / EP LYSATE and PHENOXYETHANOL and SODIUM HYDROXIDE and CITRIC ACID, phenoxyethanol can be replaced by another preservative.

This lysate comprises a content of Lactobacillus rhamnosus ranging from 0.1 to 10%, in particular from 0.5 to 5% by weight and in particular from 1 to 2% by weight of dry matter (active material) relative to the total weight of said lysate.

The L. rhamnosus lysate of the invention can be formulated in a composition at a rate of at least 0.0001% (expressed in dry weight or weight of active material), in particular at a rate of 0.0001 to 10% and more particularly at the rate of 0.0001 to 5% by weight, in particular from 0.0001 to 2% by weight, and in particular from 0.0001% to 1%, or even from 0.0001 to 0.05% by weight relative to the total weight of the composition.

According to a particular mode, the extract of Lactobacillus rhamnosus (CNCM 1-5313) is present in a content ranging from 0.0001 to 0.05% by weight of active material relative to the total weight of said composition.

The L rhamnosus lysate according to the invention can be used alone or in combination with other probiotic microorganisms, prebiotic ingredients, symbiotic ingredients or their mixture.

According to a particular embodiment, the composition also comprises at least cosmetic active agent other than vitamins or extracts containing it, in particular chosen from oils rich in essential fatty acids or not, prebiotic extracts (which facilitate the growth of certain bacterial species beneficial to the cutaneous microflora to the detriment of undesirable species), other extracts from other strains of probiotic microorganisms or even symbiotic ingredients (probiotic extract + prebiotic extracts), seboregulators (to promote the biodiversity of the flora) and firming agents (to limit the enlargement of pores), soothing agents, protective active agents in particular stimulators of antioxidant and detoxifying defenses of the skin (otherwise called antioxidant agents or simply antioxidants), depigmenting agents, exfoliating agents, antiaging active ingredients, moisturizing ingredients, immunomodulatory active ingredients , and mixtures thereof.

By "composition" according to the invention means a cosmetic or dermatological composition.

According to a particular mode, it will be a cosmetic composition. According to another particular mode, it will be a dermatological composition.

The composition according to the invention can, for example, be used as a care and / or makeup product for the face and / or the body.

The compositions according to the invention are intended more particularly for topical application to keratin materials, in particular to the skin.

The compositions of the invention comprise a physiologically acceptable medium, that is to say compatible with the skin and the integuments. The compositions may have all cosmetic forms, and in particular may be in the form of an oil-in-water or water-in-oil emulsion or multiple emulsion, a microemulsion, a solution, a suspension, '' a lotion, gel, cream, milk, serum, mist (sprayed fluid solution), stick or powder and suitable for application on the skin, the lips and / or the integuments.

According to a particular embodiment, the composition of the invention is intended for topical application to the skin and / or the lips and is in the form of a cosmetic composition for caring for and / or making up the skin and / or lips.

The cosmetic composition of the invention is advantageously in the form of an emulsion, a serum, or a mist.

The composition generally comprises at least one aqueous phase and at least one oily phase.

The aqueous phase generally represents from 1 to 99% by weight, relative to the total weight of said composition.

An oily phase according to the invention can comprise hydrocarbon oils, silicone oils, fluorinated or not, and mixtures thereof. These oils can be volatile or non-volatile, vegetable, mineral or synthetic.

Advantageously, use will be made of hydrocarbon oils, in particular of vegetable origin. As volatile hydrocarbon oils, mention may be made in particular of branched C8-C16 alkanes, branched C8-C16 esters and mixtures thereof.

As non-volatile hydrocarbon oils, mention may in particular be made of hydrocarbon oils, hydrocarbon oils of vegetable origin, synthetic ethers of C10-C40, synthetic esters of C10-C40, fatty alcohols of C12-C26, acids higher C12-C22 fats, and mixtures thereof. The oils may be present in the composition of the invention in a content ranging from 1 to 95% by weight relative to the total weight of the composition.

The composition of the invention can also comprise any additive usually used in cosmetics such as UV filters, antioxidants, surfactants, gelling agents, fillers, coloring matters (eg pigments), preservatives, film-forming polymers, perfumes, active cosmetic agents, such as, for example, emollients, moisturizers, vitamins, anti-aging agents, lightening agents, and mixtures thereof.

Cosmetic process and other uses

The invention also relates to a non-therapeutic cosmetic process intended to prevent and / or reduce the signs of skin aging and / or to promote and / or improve the barrier function comprising the application to keratin materials of at least one extract of Lactobacillus rhamnosus (CNCM 1-5313) in particular a lysate of Lactobacillus rhamnosus (CNCM 1-5313), one of its fractions and / or one of its metabolites, or of a composition according to the invention, such as previously defined.

Preferably, the keratin materials according to the invention are healthy keratin materials.

The extract of Lactobacillus rhamnosus is generally present in a content ranging from 0.0001 to 10% by weight of dry matter, in particular from 0.0001 to 5%, preferably from 0.0001 to 2%, and in particular from 0 .0001% to 1%, or even from 0.0001 to 0.05% by weight of dry matter relative to the total weight of the composition.

The composition used in the cosmetic process can be a care and / or makeup composition, in particular a care composition for the skin of the face and / or the body.

In particular, the method is intended to prevent and / or reduce the loss of firmness and density of the skin, prevent and / or reduce the appearance of wrinkles and / or fine lines and / or pigment spots, improve the radiance and / or the homogeneity of the skin, promoting and / or improving the hydration of the skin, detoxifying the skin, promoting and / or stimulating its antioxidant defenses and / or improving the resistance to hypoxia of the skin, and / or control the microflora of the skin, and / or stimulate the immunity of the skin and the formation of the skin barrier. By "promoting and / or stimulating the antioxidant defenses of keratinous materials" is meant in particular promoting oxygenation of the skin and thus reducing the effects of cutaneous hypoxia, promoting cellular respiration and the production of energy at the cellular level.

By "promoting and / or stimulating the immunity of the skin", it is intended to maintain the balance of the microflora of the skin and to promote cutaneous homestasis for healthier skin that is better protected against external agents.

According to a particular mode, the process is intended to prevent and / or reduce the loss of firmness and density of the skin, prevent and / or reduce the appearance of wrinkles and / or fine lines and / or pigment spots, improve the radiance and / or homogeneity of the skin, promoting and / or improving the hydration of the skin, and / or improving the resistance to hypoxia of the skin, and / or controlling the microflora of the skin.

According to a particular mode, the composition of the invention is applied to aged, dull, tired skin, or affected by an unfavorable or unbalanced nutritional context, emotional stress or lack of sleep.

The present invention also relates to the non-therapeutic cosmetic use of an extract of Lactobacillus rhamnosus (CNCM 1-5313) for promoting and / or improving the barrier function and / or reducing the signs of skin aging, in particular preventing and / or reduce the loss of firmness and density of the skin, prevent and / or reduce the appearance of wrinkles and / or fine lines and / or pigment spots, improve the radiance and / or uniformity of the skin, promote and / or improve the hydration of the skin, detoxify the skin, stimulate its antioxidant defenses and / or improve the resistance to hypoxia of the skin, and / or control the microflora of the skin, and / or stimulate the immunity of the skin and the formation of the skin barrier.

According to a particular embodiment, the extract according to the invention is used to promote and / or improve the barrier function and / or reduce the signs of skin aging, in particular preventing and / or reducing the loss of firmness and density of the skin , prevent and / or reduce the appearance of wrinkles and / or fine lines and / or pigmentation spots, improve the radiance and / or homogeneity of the skin, promote and / or improve skin hydration, detoxify the skin and / or improve resistance to hypoxia of the skin, and / or control the microflora of the skin. The invention also relates to an extract of Lactobacillus rhamnosus CNCM 1-5313 according to the invention for promoting and / or stimulating the immunity of the skin.

The invention will now be illustrated in the following nonlimiting examples.

Unless otherwise indicated, the percentages are weight percentages expressed by weight relative to the total weight of the composition.

EXAMPLES

The Applicant has evaluated the effect of a lysate of L. rhamnosus probiotic bacterial strain: ATCC 7469, CCM 1825, NCDO 243, NCIB 6375 or WDCM 00101 according to the collections in which it has been deposited.

In particular, the probiotic bacterial strain used was deposited according to the Budapest Treaty with the National Collection of Cultures of Microorganisms (CNCM) at the Institut Pasteur (28 rue du Docteur Roux, F-75024 Paris cedex 15) on 1 1 April 2108 in the name of BIOVITIS, Le Bourg, 15400 SAINT -ETIENNE-DE-CHOMEIL, under the designation CNCM I- 5313.

A lysate of this L rhamnosus strain is obtained according to the following protocol from the supplier Greentech:

1. Culture of the L. rhamnosus strain in an appropriate culture medium;

2. Biomass recovery by centrifugation;

3. Cell lysis (addition of soda);

4. Adjustment of the pH (addition of citric acid) and of the dry matter (addition of sterile water) to obtain an active matter content ranging from 0.01 to 10% by weight of dry matter (active matter) relative to the total weight of said lysate;

5. Possible addition of preservative (ex: phenoxyethanol).

Such a L rhamnosus lysate is called Probiophyte LB by the supplier Greentech, and defined in INCI name under the name WATER and LACTOBACILLUS FERMENT LYSATE and PHENOXYETHANOL and SODIUM HYDROXIDE and CITRIC ACID, phenoxyethanol being able to be replaced by another preservative.

This lysate comprises a content of Lactobacillus rhamnosus ranging from 0.1 to 10%, in particular from 0.5 to 5% by weight and in particular from 1 to 2%, or even from 1.75% by weight of dry matter (active material ) relative to the total weight of said lysate.

Such a lysate (but without phenoxyethanol preservative) is tested at a content of 0.05, 0.1 and 0.2% in tests on cell cultures of skin cells and used in

RECTIFIED SHEET (RULE 91) ISA / EP cosmetic compositions for topical application illustrated below in contents ranging from

I to 2% by weight of raw material.

It is interchangeably named LB Probiophyte or Lactobacillus rhamnosus lysate in the following examples.

Example 1 Effect of the extract of L rhamnosus on cultures of keratinocytes

The test implemented here made it possible to compare two extracts of probiotic bacterial strains in order to select the extract most effective in stimulating skin cells. We used an RT-qPCR methodology which allows us to observe stimulation or inhibition or the absence of stimulation of genes involved in key skin care processes, in particular on keratinocytes, the major cells of the epidermis.

Normal human keratinocytes (KHN are cultured and then treated with the probiotic extract of L rhamnosus described above or the extract of Lactococcus lactis.

After treatment of the KHNs, a study by qPCR (polymerase chain reaction) on the genes studied is carried out from the cDNAs obtained after reverse transcription of the total RNAs extracted.

Cells used and culture conditions:

Cells: normal human epidermal keratinocytes on the 3rd pass (NHEK)

Culture medium: Keratinocyte-SFM supplemented with EGF 0.25 ng / ml, pituitary extract 25 pg / ml and gentamycin 25 pg / ml at 37 ° C and with 5% C02

Test medium: Keratinocyte-SFM supplemented with gentamycin 25 pg / ml

Compounds tested:

Probiophyte LB without preservative: extract of Lactobacillus rhamnosus CNCM I- 5313 according to the invention

Lactococcus lactis extract: Lactococcus lactis extract supplied by Greentech (product ref 001 1 19, batch no LYCC1204E1 -BY of March 2012) and comprising a lactate of Lactococcus lactis, citric acid and sodium hydroxide. The dry (active) content of L lactis is 5 to 6% by weight of raw material.

Doses tested: 0.05; 0.1 and 0.2%

Processing time: 24h

N = 3 cultures by experimental conditions Analyzes performed:

Measurement of gene expression by RT-qPCR on messenger RNAs extracted from untreated cell mats (controls) and treated with the different concentrations of probiotic extract. Several genes have been evaluated; are presented in the tables below the corresponding genes and results for which differential results have been observed between the extract according to the invention (Probiophyte LB: extract of Lactobacillus rhamnosus) and the comparative extract (extract of Lactococcus lactis).

RT -gPCR technology (quantitative RT-PCR)

• Obtaining total RNA

The total RNA of each sample was extracted using TriPure Isolation Reagent® according to the protocol recommended by the supplier. The quantity and quality of RNA were evaluated by capillary electrophoresis (Bioanalyzer 2100, Agilent).

Traces of potentially contaminating DNA were removed by treatment with the DNA-free system (Ambion).

• Synthesis of complementary DNAs

Complementary DNAs (cDNAs) were synthesized by reverse transcription of total RNAs in the presence of oligo (dT) and the enzyme "Transcriptor First strand cDNA synthesis" (Roche Diagnostics). The cDNA obtained was quantified by spectrophotometry (Nanovue; GE Healthcare), then the cDNA quantities were adjusted to 5 ng / mI.

• Quantitative PCR

The PCR reactions (polymerase chain reactions) were carried out by quantitative PCR (Light Cycler; Roche Molecular Systems Inc.) and according to the procedures recommended by the supplier.

The reaction mixture (10 μl final) for each sample contained:

2.5 mI of cDNA at 5 ng / mI,

the primers of the different markers used,

the reaction mixture containing the taq DNA polymerase enzyme, the SYBR Green marker

Traitem

The raw data was transferred and processed under Microsoft Excel ^® software. The incorporation of fluorescence into the amplified DNA is measured continuously during the PCR cycles. These measurements make it possible to obtain fluorescence intensity curves as a function of the PCR cycles and thus to evaluate a relative expression value (ER) for each marker.

The number of cycles is determined from the "exit" points of the fluorescence curves. For the same marker analyzed, the later a sample leaves (high number of cycles), the lower the initial number of copies of the mRNA.

The value of ER (relative expression) is expressed in arbitrary units (AU) according to the following formula: 1/2 ^{number of} cycles) x 10 ⁶

Table 1:

Genes studied Table 2:

Results showing the stimulating effect of the LB probiophyte according to the invention on several keratinocyte genes compared to the extract of Lactococcus lactis.

The table above shows the stimulating effect of Probiophyte LB on several important skin processes:

the formation of the skin barrier with the stimulation of the transglutaminase gene (TGM1) which allows the formation of the corneal envelopes of the corneocytes which ensure the mechanical and chemical resistance and the hydrophobicity of the surface of the skin as well as the organization of the lipids of the surface thereof;

innate immunity with the stimulation of several genes of antimicrobial peptides which make it possible to control the development of the cutaneous microflora: DEFB4A, S100A7, PI3, RNASE7, and

strengthening the dermo-epidermal junction with the stimulation of the collagen type 7 gene (COL7A1), a collagen of cohesion between the dermis and the epidermal junction. This collagen decreases during aging.

Example 2 Effect of the extract of L rhamnosus on cultures of keratinocytes, melanocytes and fibroblasts

This second experiment makes it possible to deepen the biological effects of the LB Probiophyte, using the TLDA (TaqMan Low Density Array) methodology on a larger gene repertoire and on the three main cell types of the skin: keratinocytes, melanocytes, and fibroblasts.

Cells used:

Cells: 5 ^th normal human epidermal keratinocytes (KHN), 6 ^th normal human dermis ^fibroblasts , 7 ^th normal human melanocytes (MHN)

Compounds tested:

Probiophyte LB without preservative): extract of Lactobacillus rhamnosus CNCM 1-5313

Doses tested: 0.1 and 0.2 pg / ml

Processing time: 24h

N = 3 cultures by experimental conditions

Analyzes performed:

Measurement of gene expression by RT-qPCR from the messenger RNAs extracted from untreated cell mats (controls) and treated with the different concentrations of Probiophyte LB.

TLDA technology (TaqMan Low Density Array)

• Obtaining total RNA The cell culture medium is eliminated, and 250 μL of RLT lysis buffer (supplied in the Nucleospin RNA trace kit, Macherey-Nagel) are added. The cells are scraped using a Cell Sera per then, the cell lysate is recovered in a 1.2 ml deepwell (supplied in the Nucleospin RNA kit). Total RNAs are extracted according to defined protocols.

The total RNA solutions obtained are assayed, and their quality verified, using a microplate reader, spectrostarNANO (BMG Labtech) coupled to MicrolabSTAR. This device is connected to the computer controlling the robotic platform and has specific software for analyzing the results (MARS software). The technique requires a 384-well microplate (LoBase), a positive control (RNA 250, AM7155, Thermofisher) to validate the pipetting performed by the robot as well as the values generated by the spectrostarNANO reader.

• Synthesis of complementary DNAs

The reverse transcription kit (RT) used is the High Capacity Reverse Transcription Kit (Thermo Fisher). It was used according to the protocol provided. 500 ng of total RNA are diluted in water for a final volume of 25 pL. They are then incubated for 10 minutes at 25 ° C and then 2 hours at 37 ° C in the presence of 25 μL of reaction mixture of High Capacity Reverse Transcription Kit 2X previously prepared as indicated below. The different incubations are done within TRobot (Biométra).

Table 3:

High Capacity Reverse Transcription Kit 2X reaction mixture for 1 reaction

• PCR-TaqMan Low Density Array

15 μL of each RT is mixed with 60 ml of water and then 75 μL of TaqMan Gene Expression master mix (ThermoFisher), containing the DNA polymerase, are added. After homogenization, 100 μL are deposited on the microfluidic cards containing the probes corresponding to the genes tested, the latter are centrifuged and then sealed. The CD Rom corresponding to the profile of the genes deposited on the plates is loaded into the SDS 2.3 software, specifying the location of each gene on the map. The control gene (or “endogenous gene”) to be used for the normalization of the results must be indicated before the launch of the PCR. The latter is carried out according to the protocol provided by Applied Biosystems in the ABI Prism 7900HT Sequence detection System. The qPCR steps are 2 min at 50 ° C, 10 min at 94.5 ° C then 30 s at 97 ° C and 1 min at 59.7 ° C for 40 cycles.

Statistical analyzes

Quantitative real-time PCR can be used if its efficiency is between 90% and 1 10%. For each sample, the number of cycles at which the signal appears is determined by the SDS 2.3 software. For the same test, the expression levels of the transcripts of interest obtained are normalized with respect to the value obtained for the household gene Beta-2-microglobulin. This gene, the expression of which is constitutive and invariant, makes it possible to overcome any variations induced during the experiment (assay of total RNAs, pipetting, reverse transcription step, PCR in the apparatus).

In the RT-PCR TLDA method, quantification is performed using the comparative method of AACt. The relative quantification values (RQ) obtained correspond to the amplitude level (x times more or less than the control) of the expression compared to our control here the non-irradiated. The RQ is obtained by the following calculation where the control is equal to 1: Q = 2 ^DD = 2 (^ ^t treated - ACt not treated)

ACt treated = Ct target gene treated - Ct household gene treated

Untreated ACt Untreated target gene Ct - Untreated household gene Ct

In order to assess statistically significant variations in transcriptional activity, we will use Student's t-test. Each condition is performed in triplicate (3 untreated and 3 treated under the same conditions). Fischer's F-test is first applied by comparing the two data matrices. When the value is greater than a = 0.05 then the variance for Student's t test is 2, when the Fischer F-test is less than a =

0.05 then the variance will be equal to 3. The transcriptional variations retained will be those with a Student's t test less than a = 0.05.

The results are presented on average over n = 3. Student's t-test was used to compare the effect between treated and untreated cells.

The results are considered significant for p <0.05 (*) or p <0.01 (**).

Below are the results tables showing the effect of Probiophyte LB on several keratinocyte genes (KHN) complementary to those of the previous study, and on several melanocytic (MHN) and fibroblastic (FHN) genes. Values less than 1 correspond to an inhibiting effect on the expression of said genes and values greater than 1 correspond to a stimulating effect on the expression of said genes. Table 4:

Results on the gene expression of melanocytes

Table 5:

Results on gene expression of keratinocytes

Table 6

Results on fibroblast gene expression

These results show that dermal fibroblasts are much more sensitive to the modulation of their genes by the LB Probiophyte (n = 22 modulated genes) than are keratinocytes (n = 2 modulated genes) and melanocytes (n = 1 modulated gene) ). The extract therefore has a very important activity in the cells of the dermis.

For dermal fibroblasts, activation, in particular, of the genes involved in: Lactobacillus rhamnosus lysate has been observed.

the attachment and cell-matrix cohesion which allows the fibroblast to densify and contract the matrix with the positive effects of this process on firmness (ITGB1);

maintaining the integrity of cellular structures such as the nucleus and therefore the resistance of skin cells (SYNE2, LMNA);

the formation of several collagens and elastin which are the major constituents of the dermis and whose stimulation is opposed to the effects of aging (COL1A1, COL5A1, COL13A1, ELN);

the water balance of the dermis, whether it is water movements (AQP1) or water reserves with the formation (HAS2 and 3), fixation (CD44) and secretion (ABCC5) of hyaluronic acid which can capture up to 1000 times its weight in water;

regeneration of dermal cells (Ki67);

antioxidant defenses of cells (GPX1) and detoxifying (ABCC1 = MRP1). This last gene codes for a protein which will allow compounds that may be toxic to it to be expelled from the cell;

the adaptation of cells to hypoxia conditions (HIF1a) which increase with age in the skin (cf. markers of hypoxia such as anhydrase carbonic 9 in aged skin) and which can be explained by the reduction of the dermal vascular network with age;

energy and nutrition with the entry of glucose into cells (SLC2A1);

energy production in the mitochondria (MT = NADH dehydrogenase or ubiquinone);

detoxification and therefore protection of the mitochondria through the degradation and recycling of damaged mitochondrial proteins (LONP1).

For keratinocytes we observed:

activation of a gene involved in innate immunity (TLR1) and which is an immune monitoring receptor to bacterial constituents of the lipopeptide type

inhibition of a gene involved in the breakdown of collagens (MMP9), a process that increases with aging.

For melanocytes, we observed:

a drop in a factor stimulating melanogenesis in these cells (PPARG) which contributes to a depigmenting effect.

All of these results therefore show that the extract of Lactobacillus rhamnosus CNCM 1-5313 according to the invention applied to keratin materials, in particular the skin, makes it possible to prevent and / or reduce the loss of firmness and density of the skin, prevent and / or reduce the appearance of wrinkles and / or fine lines and / or pigmentation spots, improve the radiance and / or consistency of the skin, promote and / or improve skin hydration, detoxify the skin, stimulating its antioxidant defenses and / or improving the resistance to hypoxia of the skin, and / or controlling the microflora of the skin, and / or stimulating the immunity of the skin and the formation of the skin barrier.

Example 3: Formulations

The following formulations are prepared according to conventional methods of formulation in the cosmetic field.

Anti-aging regenerating serum

Water qs 100% Alcohol 5%

Glycols 8%

Isotridecyle isononanoate 4%

Squalane 2%

2% caprylic / capric acid triglyceride

Methyl gluceth-20: 2%

Steareth-2: 0.5%

Steareth-21: 0.5%

Carbomer 0.3%

0.2% sodium hyaluronate

0.1% xanthan gum

Sodium hydroxide 0.1%

Adenosine 0.04%

L rhamnosus lysate 2%

Applied to the skin, the composition makes it possible to improve the firmness of the skin and reduce wrinkles.

Purifying mask

Water qs 100%

Kaolin 18%

Stearic acid 3.5%

Palmitic acid 3.5%

Glycols 6%

Titanium dioxide 3%

Lauric acid 2%

Glyceryl stearate 1.5%

PEG-100 stearate 1.5%

Potassium hydroxide 1, 4%

Phenoxyethanol 0.7%

0.5% acrylic polymer

0.05% xanthan gum

L rhamnosus lysate 1%

Zinc gluconate 0.05% Applied to the skin, the composition makes it possible to reduce the rate of sebum, reduce the size of the pores, improve the skin texture.

Anti-pollution detoxifying lotion

Water qs 100%

Glycols 15%

Alcohol 5%

Phenoxyethanol 0.8%

PEG-60 hydrogenated castor oil 0.5%

Sodium hyaluronate 0.1%

L rhamnosus lysate 0.5%

0.5% prebiotic extract

Applied to the skin, the composition makes it possible to give the skin a healthy glow, a radiant, even complexion.

Whitening Essence

Water qs 100%

Glycols 1 1%

Propanediol 5%

Dimethicone 2%

Alcohol 1%

Tromethamine 1.5%

Phenoxyethanol 0.9%

0.6% acrylic copolymer

Sodium citrate 0.5%

L rhamnosus lysate 2%

Ascorbyl glucoside (vitamin CG) 0.5%

Applied to the skin, the composition makes it possible to improve the uniformity of the complexion.

Radiance foundation

Water qs 100%

Hydrocarbon oils 12%

Titanium dioxide 10% Propanediol 5%

Glycerin 5%

Zinc oxide 3%

Surfactants 3%

1% film-forming polymer

Potassium sorbate 0.1%

L rhamnosus lysate 1%

Applied to the skin, the composition makes it possible to impart a healthy glow effect, a radiant, even complexion.

Vitamin serum

Glycerol 4%

Butylene glycol 3%

Carbomer 0.5%

Lactic acid 1%

L rhamnosus lysate 2%

Sodium hydroxide 0.7%

0.1% xanthan gum

0.2% acrylic gelling agent

Alcohol 3%

Ascorbyl glucoside (vitamin CG) 0.5%

Phenoxyethanol 0.5%

Dyes qs%

Water qs100%

Applied to the skin, the composition improves the firmness of the skin and gives it a healthy glow effect.

Mist

Glycerol 2%

Glycol s 4%

Gelling agents 2%

L rhamnosus lysate 1%

0.25% caprylic / capric acid triclyceride Emulsifiers 0.6%

Phenoxyethanol 0.5%

Tocopherol (vitamin E) 0.02%

Water qs 100%

Applied to the skin, the composition makes it possible to promote and / or improve the nutrition of the skin and give it a healthy glow effect, a radiant and homogeneous complexion.

Pct

(original in electronic form)

RESERVED AT THE RECEIVING OFFICE

RESERVED AT THE INTERNATIONAL OFFICE

SUBSTITUTE SHEET (RULE 26)

Claims

1. Composition for topical application to keratin materials, in particular the skin and / or the lips, comprising, in a physiologically acceptable medium, at least one extract of Lactobacillus rhamnosus CNCM 1-5313, of one of its fractions and / or of one of its metabolites and at least one excipient chosen from solvents other than water, surfactants, gelling agents, thickeners, fillers, coloring matters, film-forming polymers, perfumes and their mixtures, characterized in that it is a Lactobacillus rhamnosus CNCM 1-5313 lysate, one of its fractions and / or one of its metabolites.

2. Composition according to claim 1, characterized in that the lactate of Lactobacillus rhamnosus CNCM 1-5313 comprises from 0.1 to 10%, in particular from 0.5 to 5% and in particular from 1 to 2% by weight in matter (s) dry (s) (active) relative to its total weight.

3. Composition according to claim 1 or claim 2, characterized in that the extract of Lactobacillus rhamnosus CNCM 1-5313 is present in a content ranging from 0.0001 to 10%, in particular from 0.0001 to 5%, in particular from 0.0001 to 2% by weight, and in particular from 0.0001% to 1%, or even from 0.0001 to 0.05% by weight of active material (dry matter) relative to the total weight of said composition.

4. Composition according to any one of claims 1 to 3, characterized in that it further comprises at least cosmetic active other than vitamins or extracts containing it, in particular chosen from oils rich in essential fatty acids or not , prebiotic extracts, other extracts from other strains of probiotic microorganisms or even symbiotic ingredients, seboregulators and firming agents, soothing agents, antioxidants, depigmenting agents, exfoliating agents, antiaging active ingredients, ingredients moisturizers, immunomodulatory active ingredients, and mixtures thereof.

5. Composition according to any one of claims 1 to 4, characterized in that it is in the form of an oil-in-water or water-in-oil emulsion or of a multiple emulsion, of a solution , a suspension, a lotion, a gel, a cream, a milk, a serum, a mist, a stick or a powder.

6. Composition according to any one of claims 1 to 5, characterized in that it is a cosmetic composition for caring for and / or making up the skin and / or the lips.

7. Non-therapeutic cosmetic process intended to prevent and / or reduce the signs of skin aging and / or to promote and / or improve the barrier function, comprising the application to keratin materials of at least one extract of Lactobacillus rhamnosus CNCM 1 -5313, in particular a lactate of Lactobacillus rhamnosus CNCM 1-5313, of one of its fractions and / or of one of its metabolites, or of a composition as defined in any one of Claims 1 to 6.

8. Cosmetic method according to claim 7, characterized in that it is intended to prevent and / or reduce the loss of firmness and / or density of the skin, prevent and / or reduce the appearance of wrinkles and / or fine lines and / or pigment spots, improve the radiance and / or uniformity of the skin, promote and / or improve the hydration of the skin, detoxify the skin, and / or improve the resistance to hypoxia of the skin , and / or control the microflora of the skin.

9. Cosmetic method according to claim 7 or claim 8, characterized in that the composition is applied to aged, dull, tired skin, or impacted by an unfavorable or unbalanced nutritional context, emotional stress or lack of sleep.

10. Non-therapeutic cosmetic use of an extract of Lactobacillus rhamnosus CNCM 1-5313, in particular a lysate of Lactobacillus rhamnosus CNCM 1-5313, of one of its fractions and / or of one of its metabolites, for promoting and / or improve the barrier function and / or reduce the signs of skin aging, in particular prevent and / or reduce the loss of firmness and / or skin density, prevent and / or reduce the appearance of wrinkles and / or fine lines and / or pigment spots, improve the radiance and / or uniformity of the skin, promote and / or improve hydration of the skin, detoxifying the skin, and / or improving the resistance to hypoxia of the skin, and / or controlling the microflora of the skin.